Single ventricle congenital heart disease (SV) is the leading cause of cardiovascular death and indication for heart transplantation in infancy. SV comprises a spectrum of cyanotic congenital cardiac malformations that are defined by hypoxia and a univentricular circulation. These defects are universally fatal without intervention and despite advances in medical and surgical therapies, the 1-year survival for SV in the current era is only 68.7%. While the single ventricle can be a morphologic right ventricle (RV), left ventricle (LV) or of mixed morphology, it is the single RV lesions that have the worst outcome, presumably due to inherent limitations in the RV's ability to tolerate increased afterload. When a single RV serves as the sole pump to both the systemic and pulmonary circulations, not surprisingly, failure of the RV is both a common cause of death and indication for heart transplant in these patients. There are currently no proven therapies for SV heart failure and identification of targeted therapies specific to the failing SV are needed in order to improve outcomes. Phosphodiesterase-5 inhibitors (PDE5i), such as sildenafil, are used for the treatment of primary pulmonary hypertension in children due to their proven vasodilatory effects. Over the past few years, use of PDE5i in those with SV HF has increased dramatically with the intention of lowering pulmonary vascular resistance, increasing pulmonary venous return to the heart and subsequently improving cardiac output. Sildenafil has been associated with improved hemodynamics, exercise tolerance and myocardial function by echo in small series of SV patients. While the stated target of therapy in SV patients is the pulmonary vascular bed, there is increasing evidence in adults and animal models of HF that PDE5i has beneficial myocardial remodeling and functional effects. Whether the beneficial effects of PDE5i in the SV population specifically are related to afterload reduction of the failing SV, direct myocardial effects or a combination of both is unknown. The central hypothesis of this proposal is that PDE5i has direct myocardial effects in SV that result in augmented cardiac function, effects on cGMP-regulated signaling pathways and altered sarcomeric protein phosphorylation. Due to difficulties performing research in the vulnerable pediatric population in vivo, the current application utilize an ongoing explanted human heart tissue bank and a cell culture model to address the specific aims, thereby minimizing risk to children. We will analyze the effect of PDE5i on myofibril mechanics and sarcomere phosphorylation as well as force generation in trabeculae from explanted SV hearts. The current application will be the first to determine myocardial effects of PDE5i in explanted pediatric SV hearts and begin to correlate molecular findings with function. Finally, we will use pharmacologic and genetic methods to investigate the intracellular effects of PDE5i in neonatal rat ventricular myocytes (NRVMs).